全世界约有六分之一的育龄人口受不孕症影响,不仅给生育问题家庭带来治疗的经济负担,而且给患者带来心理压力,给社会和经济发展带来挑战。过早卵巢功能不全(POI)是指在40岁之前由于卵泡耗尽或剩余卵泡质量下降而导致的卵巢功能丧失,构成女性不孕症的重要原因。近年来,随着基因测序技术的快速发展,已经证明遗传因素在POI的发病中起着至关重要的作用。在患有POI的人群中,遗传研究表明,涉及减数分裂等过程的基因,DNA损伤修复,有丝分裂约占所有致病和潜在致病基因的37.4%。FA互补组M(FANCM)是一组参与DNA链间交联(ICLs)损伤修复的基因,包括FANCA-FANCW.FANCM基因的异常与女性不育有关,FANCM基因敲除小鼠也表现出与POI相似的表型。在POI患者的基因筛查中,这项研究在FANCM中发现了一个可疑变异。本研究旨在探讨FA通路的FANCM基因及其变异在POI发展中的致病机制。我们希望帮助阐明受影响个体的潜在诊断和治疗策略。
■研究中包括一名POI患者。POI患者的纳入标准如下:40岁以下的女性表现出两个或两个以上的基础血清促卵泡激素水平>25IU/L(在测试之间的最小间隔为4周),伴随着月经紊乱的临床症状,正常染色体核型分析结果,并排除其他可能导致卵巢功能障碍的已知疾病。我们对POI患者进行了全外显子组测序,并通过根据美国医学遗传学和基因组学学院(ACMG)建立的标准和指南对变异进行分类来鉴定致病基因。随后,鉴定出的变异体通过Sanger测序进行验证,并进行生物信息学分析.构建含有野生型和突变型FANCM基因的质粒并将其导入293T细胞。用野生型和突变型人FANCM质粒和pEGFP-C1空载体质粒转染的293T细胞被指定为EGFPFANCM-WT组,EGFPFANCM-MUT组,和EGFP组,分别。为了验证截短蛋白质的生产,转染后48小时从三组中提取细胞蛋白并用GFP抗体确认。为了研究对DNA损伤修复的影响,在EGFPFANCM-WT组和EGFPFANCM-MUT组转染后48小时进行免疫荧光实验,以检查变体是否影响FANCM定位在染色质上的能力.在EGFPFANCM-WT组和EGFPFANCM-MUT组均使用丝裂霉素C体外诱导ICL损伤,随后使用γ-H2AX抗体验证其对ICL损伤修复的影响。
■在来自近亲家庭的POI患者中,我们在FANCM基因中发现了一个纯合变异体,c.1152-1155del:p。Leu386Valfs*10。患者出现原发性不孕症,自16岁初潮以来经历月经不调。激素评估显示FSH水平为26.79IU/L,抗苗勒管激素(AMH)水平为0.07ng/mL。阴道超声提示两侧卵巢可视化不理想,子宫发育不良。病人的父母是一对近亲,母亲有规律的月经周期。病人有两个姐妹,其中一人死于骨肉瘤,而另一个表现出月经不调,被诊断为卵巢功能不全,仍然没有孩子。生物信息学分析显示患者FANCM基因外显子6中四个核苷酸(c.1152-1155del)缺失。该变体导致密码子386处的移码,在密码子396处引入过早终止密码子,这最终导致产生由395个氨基酸组成的截短蛋白质。体外实验表明,该变体导致产生约43kDa的截短FANCM蛋白,并导致其核定位缺陷,蛋白质只存在于细胞质中。用丝裂霉素C治疗后,突变质粒转染的293T细胞中γ-H2AX水平显著升高(P<0.01),表明该变体引起的DNA损伤修复能力的统计学显着损害。
■FANCM基因中的纯合变体,c.1152-1155del:p。Leu386Valfs*10,导致产生截短的FANCM蛋白。这种截短导致其与MHF1-MHF2复合物的相互作用位点丢失,防止其进入细胞核并随后识别DNA损伤。因此,FA核心复合物在染色质上的定位被破坏,阻碍FA途径的正常激活并降低细胞修复受损ICL的能力。通过破坏原始生殖细胞的快速增殖和减数分裂过程,卵母细胞的储备被耗尽,从而引发女性卵巢早衰。
UNASSIGNED: Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M (FANCM) is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including FANCA-FANCW. Abnormalities in the FANCM genes are associated with female infertility and FANCM gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in FANCM. This study aims to explore the pathogenic mechanisms of the FANCM genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.
UNASSIGNED: One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant FANCM genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group, the EGFP FANCM-MUT group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM\'s ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.
UNASSIGNED: In a POI patient from a consanguineous family, we identified a homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient\'s parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient\'s FANCM gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. In vitro experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (P<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant.
UNASSIGNED: The homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell\'s ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.